Casein kinase 1 belongs to serine/threonine kinase (which phosphorylates a tyrosine residue in some cases). As its isoforms in mammals seven types of isoforms, namely, α, β, γ1, γ2, γ3, δ, and ε, have been known. It has been known that these isoforms phosphorylate various types of different substrate proteins, and that the isoforms are able to activate, inactivate, stabilize or destabilize the functions of the proteins, and thus they are associated with regulation of the functions of various types of different organisms. Mammalian casein kinase 1δ or casein kinase 1ε has, as a structure thereof, a kinase domain that is similar to those of other isoforms. However, the N-terminal and C-terminal domains thereof are different from those of other isoforms. That is to say, the C-terminal domain has a plurality of autophosphorylation sites, and it is considered to be involved in regulation of autoenzyme activity. In addition, such a kinase domain comprises a sequence assumed to be associated with nuclear translocation (NLS: nuclear location signal) and a kinesin-like domain (KHD: kinesin homology domain).
It has been known that casein kinase 1δ and casein kinase 1ε are associated with circadian rhythm disorder, that casein kinase 1δ and casein kinase 1ε, are associated with central neurodegenerative disease, and that casein kinase 1δ and casein kinase 1ε are associated with cancer. Detailed information regarding the association of these casein kinases with the pathological conditions of the above-mentioned diseases has being known in studies regarding the interaction between the casein kinase 1δ and casein kinase 1ε, and target proteins interacting with the casein kinase 1δ and casein kinase 1ε, such as substrate proteins interacting with the corresponding casein kinase 1δ and casein kinase 1ε. Specific examples of a substrate protein phosphorylated by the Casein kinase 1δ and casein kinase 1ε include a period protein (Per), a tan protein (tau), p53, and β-catenin.
Today, the core of the biological clock acting as a central generator of the circadian rhythm is considered to consist of approximately 10 types of gene interaction networks called “clock genes.” Among these 10 types of gene groups, Per 1, 2 and 3 (Period 1, 2 and 3), Cry 1 and 2 (cryptochrome 1 and 2), Bmal1 (brain and muscle ARNT-like 1), and Clock (circadian locomotor output cycles kaput) encode transcription factors. On the other hand, CK1δ and 1ε encode casein kinase 1δ and casein kinase 1ε that phosphorylate these transcription factors. It has been known that the functional abnormality of these clock genes has influence on the circadian rhythm phenotypes of various types of animals including humans. Since the molecular mechanism of such a biological clock is well conserved beyond species, it is advantageous in that the studies of clock genes can be carried out in in vitro tests regarding the abnormality of the circadian rhythm phenotypes of humans. The Clock governs a pathway for generating activation signals, among biological clock interaction networks, and activates Per, Cry and other downstream target genes. On the other hand, Per and Cry, which govern a pathway for generating regulatory signals, act to suppress the activity of the Clock. Casein kinase 1δ and casein kinase 1ε phosphorylate Per and Cry, so as to promote the cytoplasmic degradation of Per. Moreover, the results of such phosphorylation are associated with the control of the nuclear translocation of these transcription factors and the stability thereof in the nucleus. Thus, it has been considered that the rhythm of internal molecular vibrations is governed in a living body. In the case of mammals, the biological clock is present in the suprachiasmatic nucleus (SCN), and this SCM biological clock operates together with the gene expression biological clocks of central and peripheral tissues, other than SCN.
Per has been known as a circadian rhythm regulatory protein in a living body. The mRNA and protein levels of Per vibrate in response to the circadian rhythm, and are closely associated with the control of the biological clock. For instance, it has been known that, with a decrease in the phosphorylation caused by casein kinase 1ε or casein kinase 1δ, a genetic disease having a human Per2 phosphorylation site mutation (S662G) progresses to familial advanced sleep phase syndrome (FASPS). This shows that Per plays an important role in sleep regulation. It has been known that a change in the intracellular protein amount of Per is controlled by the phosphorylation caused by casein kinase 1ε or casein kinase 1δ. That is, it has been known that, if Per is phosphorylated by these kinases, the stability of the protein significantly decreases.
Xu, Y. et al. have reported that human Per2 phosphorylation site mutated (S662G) transgenic mice were found to have the same phenotype as FASPS found in humans. Moreover, these researchers have studied the influence caused by a change in the expression level of casein kinase 1δ using hybrid mice between the above-described transgenic mice and casein kinase 1δ WT mice (WT: wild type) or casein kinase 1δ+/−(heterozygous knockout) mice. As a result, the researchers have reported that the above-described phenotype has been influenced thereby, and that the abnormality of the circadian rhythm phenotype found in the wild-type mice was corrected in the +/− mice. This report describes the phosphorylation status of Per2 and the importance of the association of casein kinase 1δ with the phosphorylation (Non Patent Literature 5). Furthermore, Badula, Loi et al. have reported that the phase of circadian rhythm can be significantly delayed by subcutaneously administering to rats a casein kinase 1ε inhibitory compound, 4-[3-cyclohexyl-5-(4-fluoro-phenyl)-3H-imidazol-4-yl]-pyrimidin-2-ylamine (PF-670462) (Non Patent Literature 4). Thus, the phosphorylation status of Per has a relationship with circadian rhythm, and the inhibitor of casein kinase 1δ or casein kinase 1ε provides a novel method of adjusting such circadian rhythm. It can be anticipated that a technique of shilling or resetting the phase of circadian rhythm contributes to the treatment of circadian rhythm disorder including various types of sleep disorders.
However, conventional inhibitors including PP-670462 as a typical example exhibit inhibitory action even against kinases (e.g. p38α) that cause concerns about the expression of side effects. Thus, such conventional inhibitors have not yet been completed as pharmaceutical products.
Almost no pharmaceutical agents for directly treating circadian rhythm disorder have been known in prior art techniques. In addition, as therapeutic agents for such sleep disorders, sleep inducing drugs have been developed and used in clinical sites. On the other hand, the development of drugs for improving circadian rhythm sleep disorder (shift work sleep disorder, jet lag syndrome, advanced sleep phase syndrome, and delayed sleep phase syndrome) and the like has not yet been completed. Also, drug therapy, which is based on the technique of shifting or resetting the phase of circadian rhythm for other sleep disorders (insomnia, sleep-related breathing disorder, central hypersomnia, parasomnia, and sleep-related movement disorder), has not yet been completed.
Hereinafter, the correlation of casein kinase 1δ or casein kinase 1ε with central neurodegenerative disease, and in particular, with Alzheimer's disease, will be described.
It has been well known that aggregation of a tau protein in an Alzheimer's disease lesion site is an important marker for the pathological conditions. Also, it has been well known that excessive phosphorylation of this tau protein is deeply associated with aggregation. A casein kinase 1 family that is excessively expressed in the lesion site is considered to include candidate kinases for phosphorylating the tan protein. Thus, among these casein kinases, Li, Guibon et al. have studied casein kinase 1δ using a HEK-293 cell expression line. As a result, they have demonstrated using a nonselective casein kinase 1 inhibitory compound, 3-[(2,3,6-trimethoxy phenyl)methylidenyl]-idolin-2-one (IC261), that casein kinase 1δ first associates with a tau protein in situ and the casein kinase 1δ directly phosphorylates the tau protein, and that the phosphorylated level in the site of the tan protein that is the same as that phosphorylated in vitro is increased due to the excessive expression of the casein kinase 1δ (Non Patent Literature 6). On the other hand, Hanger, Diane P. et al, have made a comparison by mass spectrometry between, what is called, insoluble tau (PHF-tau (paired helical filaments-tau), which is an extremely phosphorylated aggregate obtained from the lesion site of an Alzheimer's disease patient, and the phosphorylation site of a healthy human, and have then identified a phosphorylation site characteristic for the lesion site of the Alzheimer's disease patient. At the same time, based on the characteristics of the phosphorylation site, they suggested that, as candidate kinases, casein kinase 1δ as well as glycogen synthase kinase 3β, is highly likely to be associated with the process of lesion development (Non Patent Literature 7).
Hereinafter, the correlation of casein kinase 1δ or casein kinase 1ε with central neurodegenerative disease, and particularly with Alzheimer's disease, will be further described.
With regard Alzheimer's disease, it has been considered that accumulation of amyloid-β (Aβ) showing toxic to nerve cells is associated with the lesion thereof. At the same time, it has been known that the expression of casein kinase 1 is increased in the lesion site of an Alzheimer's disease patient. It is considered that Aβ is formed by cleaving APP (amyloid precursor protein) with β-secretase (aspartyl protease β-secretase) and γ-secretase (presenin-dependent protease γ-secretase). Flajolet, Marc et al. have performed an in silico analysis to study a site commonly phosphorylated by casein kinases 1 that are assumed to be present in the sequences of the subunits of these APP, β-secretase and γ-secretase. Subsequently, based on the obtained results, they have attempted to excessively express casein kinase 1ε constitutively active to N2A cells (N2A-APP695 cells) that stably express APP. As a result, they have reported that the amounts of Aβ40 and Aβ42 had become approximately 2 times and 2.5 times higher than that of a control, respectively. Furthermore, they have also reported that, when a nonselective casein kinase 1 inhibitory compound IC261 was added to this system, the amounts of Aβ40 and Aβ42 were decreased, and further that the same results could be obtained also using other two different types of nonselective casein kinase 1 inhibitory compounds, CKI-7 and D4476 (Non Patent Literature 8).
These reports (Non Patent Literature 6, 7 and 8) strongly suggest that casein kinase 1, and particularly, casein kinase 1δ or casein kinase 1ε is associated with the development of Alzheimer's disease, and that Alzheimer's disease can be treated by inhibiting the activity of the above-described enzyme.
Moreover, the chromosome 21, in which an Alzheimer's disease-causing gene is assumed to be present, becomes trisomic (triploid) in the somatic cells of a Down's syndrome patient. Thus, it has been thought that Down's syndrome can be as model for the studies of the genetic background or development of Alzheimer's disease. In particular, abnormal accumulation of specific proteins, found in the two types of diseases, has been considered to be one important, pathological and biochemical indicator associated with the pathogenic mechanism thereof, and thus has been studied. As a matter of fact, it has been known that Down's syndrome patients often have Alzheimer's disease-like cerebral lesion after middle age (approximately 35 years old). These facts strongly suggest that, even regarding neurodegenerative disease associated with Down's syndrome, this disease can be treated by inhibiting the enzyme activity of casein kinase 1, and particularly, casein kinase 1δ or casein kinase 1ε.
In prior art techniques, there have been known almost no pharmaceutical agents for treating central neurodegenerative diseases including Alzheimer's disease, which involve, as a point of action, direct inhibition of the aggregation of a tau protein or amyloid β. In addition, in prior art techniques, drug therapy for impeding the progression of central neurodegenerative diseases based on the concerned mechanism has not yet been completed.
Hereinafter, the correlation of casein kinase 1δ or casein kinase 1ε with cancer, and particularly, with pancreatic cancer, will be described.
The casein kinase 1 family is associated with regulation of various important physiological activities in cells. The casein kinase 1 family phosphorylates a wide variety of substrate proteins. For example, a tumor suppressor factor p53 and an oncogene mdm2 are both important proteins for controlling canceration and, at the same time, are substrates of casein kinase 1. Depending on the phosphorylation status thereof, cell canceration is considered to be accelerated. Among isoforms of casein kinase 1, phosphorylation of p53 by casein kinase 1ε or casein kinase 1δ, a consequent change in the interaction between p53 and mdm2, and stabilization and activation of p53 have attracted a lot of attention. Furthermore, it has also been known that casein kinase 1ε or casein kinase 1δ is involved in a regulatory protein associated with the formation of a spindle as a central body during cell division, and that the casein kinase 1ε or casein kinase 1δ is involved in apoptosis by TRAIL (tumor necrosis factor-related apoptosis inducing factor) and Fas.
By the way, pancreatic ductal adenocarcinomas (PDACs) have been considered to be refractory cancers. Brockschmidt, C. et al. have studied that casein kinase 1ε or casein kinase 1δ is highly expressed in PDACs. Based on the obtained results, a nonselective casein kinase 1 inhibitory compound IC261 was added to a human pancreatic cancer cell line in vitro. As a result, suppression of the cell growth was observed. At the same time, the same pancreatic cancer cell line was transplanted into the subcutis of a mouse, and the nonselective casein kinase 1 inhibitory compound IC261 was then administered to the mouse. As a result, Brockschmidt, C. et al. have reported that a significant effect of suppressing the growth of tumor cells was obtained as in the case of a gemcitabine administration group (Non Patent Literature 9).
In prior art techniques, a pharmaceutical agent that can be used as an anticancer agent based on inhibition of casein kinase 1ε or casein kinase 1δ has not been known in the prior art. Moreover, in prior art techniques, drug therapy for treating refractory pancreatic cancer based on the concerned mechanism has not yet been completed.